Amelioration of diabetic retinopathy in db/db mice by treatment with different proportional three active ingredients from Tibetan medicine Berberis dictyophylla F.

ETHNOPHARMACOLOGICAL RELEVANCE: Berberis dictyophylla F., a famous Tibetan medicine, has been used to prevent and treat diabetic retinopathy (DR) for thousands of years in clinic. However, its underlying mechanisms remain unclear. AIM OF THE STUDY: The present study was designed to probe the synergistic protection and involved mechanisms of berberine, magnoflorine and berbamine from Berberis dictyophylla F. on the spontaneous retinal damage of db/db mice. MATERIALS AND METHODS: The 14-week spontaneous model of DR in db/db mice were randomly divided into eight groups: model group, calcium dobesilate (CaDob, 0.23 g/kg) group and groups 1-6 (different proportional three active ingredients from Berberis dictyophylla F.). All mice were intragastrically administrated for a continuous 12 weeks. Body weight and fasting blood glucose (FBG) were recorded and measured. Hematoxylin-eosin and periodic acid-Schiff (PAS) stainings were employed to evaluate the pathological changes and abnormal angiogenesis of the retina. ELISA was performed to assess the levels of IL-6, HIF-1α and VEGF in the serum. Immunofluorescent staining was applied to detect the protein levels of CD31, VEGF, p-p38, p-JNK, p-ERK and NF-κB in retina. In addition, mRNA expression levels of VEGF, Bax and Bcl-2 in the retina were monitored by qRT-PCR analysis. RESULTS: Treatment with different proportional three active ingredients exerted no significant effect on the weight, but decreased the FBG, increased the number of retinal ganglionic cells and restored internal limiting membrane. The results of PAS staining demonstrated that the drug treatment decreased the ratio of endothelial cells to pericytes while thinned the basal membrane of retinal vessels. Moreover, these different proportional active ingredients can markedly downregulate the protein levels of retinal CD31 and VEGF, and serum HIF-1α and VEGF. The gene expression of retinal VEGF was also suppressed. The levels of retinal p-p38, p-JNK and p-ERK proteins were decreased by drug treatment. Finally, drug treatment reversed the proinflammatory factors of retinal NF-κB and serum IL-6, and proapoptotic Bax gene expression, while increased antiapoptotic Bcl-2 gene expression. CONCLUSIONS: These results indicated that DR in db/db mice can be ameliorated by treatment with different proportional three active ingredients from Berberis dictyophylla F. The potential vascular protection mechanisms may be involved in inhibiting the phosphorylation of the MAPK signaling pathway, thus decreasing inflammatory and apoptotic events.

Effect of vitamin B12 supplementation on retinal lesions in diabetic rats.

Purpose: Diabetic retinopathy (DR) is the most common complication of diabetes involving microvasculature and neuronal alterations in the retina. Previously, we reported that vitamin B12 deficiency could be an independent risk factor for DR in humans. However, the effect of vitamin B12 supplementation in experimental DR is unknown. Thus, in this study, we investigated the impact of dietary supplementation of vitamin B12 on retinal changes in diabetic rats. Methods: Diabetes was induced in 2-month-old Sprague-Dawley rats and maintained for 4 months. One group of diabetic rats were fed normal levels of vitamin B12, and one group double the quantity of vitamin B12 (50 µg/kg diet). Vitamin B12 and homocysteine levels in the plasma were analyzed with radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC), respectively. At the end of 4 months of experimentation, the eyeballs were collected. Retinal changes were analyzed with hematoxylin and eosin (H&E) staining, immunoblotting, and immunofluorescence methods. Results: Dietary supplementation of vitamin B12 had no effect on food intake, bodyweight, fasting blood glucose, and plasma homocysteine levels in the diabetic rats. However, vitamin B12 supplementation prevented loss of rhodopsin, and overexpression of VEGF, and completely prevented overexpression of HIF1α, GFAP, and endoplasmic reticulum (ER) stress markers (GRP78, ATF6α, XBP1, CHOP, and caspase 12) in the diabetic rat retina. Further, vitamin B12 ameliorated apoptosis in the retina as shown with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and prevented retinal thinning. Conclusions: Vitamin B12 supplementation of diabetic rats appeared to be beneficial by circumventing retinal hypoxia, VEGF overexpression, and ER stress-mediated cell death in the retina. The present study adds another potential therapeutic strategy of vitamin B12 in diabetes.

Effect of Moxibustion on HIF-1α and VEGF Levels in Patients with Rheumatoid Arthritis.

Background: Moxibustion has a therapeutic effect of reducing swelling and relieving pain in patients with rheumatoid arthritis (RA) but its mechanism is uncertain. Objective: To evaluate the effect of moxibustion on serum levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in patients with RA and to explore the possible mechanism of moxibustion. Methods: This study involved 46 RA patients who had fulfilled the inclusion criteria and were randomly assigned to a treatment group and a control group in an equal ratio. The control group was treated with methotrexate or leflunomide, while the treatment group received methotrexate or leflunomide and moxibustion at ST 36 (Zusanli), BL 23 (Shenshu), and Ashi points. Patients' clinical symptoms, RA-associated serum markers, and serum levels of TNF-α, IL-1ß, HIF-1α, and VEGF were compared in the two groups before and after intervention. Statistical analysis was performed using SPSS 21.0 statistical software. Results: 37 of 46 RA patients eventually completed the whole treatment course. Compared with the control group, the treatment group significantly improved the clinical symptoms (P < 0.05) but with no significant differences in RA-associated serum markers (P > 0.05). There were significant differences in TNF-α and IL-1ß among the groups after 8 weeks of treatment (P < 0.05). HIF-1α and VEGF were decreased in the treatment group after therapy (P < 0.05). VEGF was reduced in the control group (P < 0.05), while HIF-1α was not significantly improved (P > 0.05). The reductions of HIF-1α and VEGF in the treatment group were superior to the control group (P < 0.05). Conclusions: Moxibustion enhanced the anti-inflammatory and analgesic effects of conventional medicine and can enhance the effect of conventional medicine, downregulating HIF-1α/VEGF contents to inhibit angiogenesis.

Amelioration of diabetic nephropathy in db/db mice treated with tibetan medicine formula Siwei Jianghuang Decoction Powder extract.

Siwei Jianghuang Decoction Powder (SWJH) documented originally in the Four Medical Tantras-Blue Glaze exhibited beneficial effects on diabetic nephropathy (DN) via combined synergistically action of multiple formula components including Curcumae longae Rhizoma, Berberidis dictyophyllae Cortex, Phyllanthi Fructus and Tribuli Fructus. This study investigated the effects of SWJH on DN in db/db mice and possible underlying mechanisms. The ten weeks old db/db mice treated with SWJH by intra-gastric administration once a day for 8 weeks. After 8 weeks, body weight, water and food intake of mice were recorded. The level of fasting blood glucose (FBG) was measured. Serum creatinine (Scr), blood urea nitrogen (BUN), urine microalbumin (UMAlb), serum uric acid (UA) and urinary albumin excretion (UAE) were detected. An enzyme-linked immunosorbent assay was performed to test serum vascular endothelial growth factor (VEGF) and transforming growth factor-ß1 (TGF-ß1). Real-time PCR and Western blot analysis were used to test mRNA and protein expression of hypoxia inducible factor-1α (HIF-1α), VEGF and TGF-ß1 in kidney tissue. SWJH treatment significantly reduced the levels of FBG, Scr, BUN, UMAlb, UA and UAE and retarded renal fibrosis. SWJH treatment further significantly reduced serum TGF-ß1 level and downregulated the expression of HIF-1α, VEGF and TGF-ß1 at both mRNA and protein levels. Principal component analysis and partial least squares regression and hierarchical cluster analysis demonstrated that SWJH treatment significantly ameliorated renal damage in DN mice. These consequences suggested that SWJH formulations were effective in the treatment of DN through regulating the HIF-1α, VEGF and TGF-ß1 overexpression.

Hyperbaric oxygen therapy improves the effect of keloid surgery and radiotherapy by reducing the recurrence rate.

OBJECTIVE: Keloids are exuberant cutaneous scars that form due to abnormal growth of fibrous tissue following an injury. The primary aim of this study was to assess the efficacy and mechanism of hyperbaric oxygen therapy (HBOT) to reduce the keloid recurrence rate after surgical excision and radiotherapy. METHODS: (1) A total of 240 patients were randomly divided into two groups. Patients in the HBOT group (O group) received HBOT after surgical excision and radiotherapy. Patients in the other group were treated with only surgical excision and radiotherapy (K group). (2) Scar tissue from recurrent patients was collected after a second operation. Hematoxylin and eosin (H&E) staining was used to observe keloid morphology. Certain inflammatory factors (interleukin-6 (IL-6), hypoxia-inducible factor-1α (HIF-1α), tumor necrosis factor-α (TNF-α), nuclear factor κB (NF-κB), and vascular endothelial growth factor (VEGF)) were measured using immunohistochemical staining. RESULTS: (1) The recurrence rate of the O group (5.97%) was significantly lower than that of the K group (14.15%), P<0.05. Moreover, patients in the O group reported greater satisfaction than those in the K group (P<0.05). (2) Compared with the recurrent scar tissue of the K group, the expression levels of the inflammatory factors were lower in the recurrent scar tissue of the O group. CONCLUSIONS: Adjunctive HBOT effectively reduces the keloid recurrence rate after surgical excision and radiotherapy by improving the oxygen level of the tissue and alleviating the inflammatory process.

Effect of Shenmai injection on cognitive function after cardiopulmonary bypass in cardiac surgical patients: a randomized controlled trial.

BACKGROUND: Postoperative cognitive dysfunction (POCD) is a common complication after cardiac surgery that influences the clinical outcomes and quality of life of patients. This study aimed to evaluate the effects of Shenmai injection (SMI) on POCD of patients who underwent cardiac valve replacement under cardiopulmonary bypass (CPB). METHODS: This prospective, randomized, controlled trial was conducted from September 2014 to January 2017. Eighty-eight patients receiving cardiac valve replacement under CPB were randomized into the control (C) or the SMI (S) group. SMI (0.6 mL/kg) was administered intravenously from the time of anesthesia induction to the beginning of CPB. Cognitive function was assessed at 3 days before surgery and 3 days, 7 days, and 1 month after surgery using the Beijing version of the Montreal Cognitive Assessment (MoCA-BJ) score. The serum levels of neuroglobin (Ngb), hypoxia-inducible factor-1α (HIF-1α), and neuron-specific enolase (NSE) were measured at 30 min after induction (T0), immediately after the endonasal temperature rewarmed to 36 °C (T1), and 1 h (T2), 6 h (T3), 24 h (T4), 48 h (T5), and 72 h (T6) after CPB. RESULTS: Compared with the baseline values at T0, the serum Ngb levels in group C were significantly decreased at T1-2 and then increased at T3-6, while the levels in group S were decreased at T1-2 and increased at T4-6, compared to group C (p < 0.05). The serum HIF-1α levels at T1-4 and the serum NSE levels at T1-6 were significantly increased in both groups (p < 0.05). The serum levels of Ngb at T3, HIF-1α at T1-3, and NSE at T3-4,6 were lower in group S, compared to group C (p < 0.01). The MoCA-BJ scores were decreased at 3 and 7 days after surgery in both groups, and the MoCA-BJ scores in group S were higher than those in group C at 3 and 7 days after surgery (p < 0.01). CONCLUSION: Cognitive function is impaired postoperatively in patients who have undergone cardiac valve replacement under CPB. In addition, treatment with the traditional Chinese medicine SMI decreases the serum levels of Ngb, HIF-1α, and NSE as well as attenuates cognitive dysfunction. TRIAL REGISTRATION: This trial was registered with Clinicaltrials.gov as ChiCTR-TRC-14004373 on March 11, 2014.

Transarterial Chemoembolization Using Sorafenib in a Rabbit VX2 Liver Tumor Model: Pharmacokinetics and Antitumor Effect.

PURPOSE: To investigate feasibility, safety, and effect of transarterial chemoembolization using sorafenib on degree of tumor necrosis in a rabbit VX2 liver tumor model. MATERIALS AND METHODS: New Zealand White rabbits (n = 20) with a VX2 tumor were divided into two groups; one group was treated with hepatic arterial administration of 0.5 mL ethiodized oil alone (Lipiodol; Guerbet, Aulnay-sous-Bois, France) (transarterial embolization with Lipiodol [TAE-L] group), and one group was treated with 0.5 mL ethiodized oil plus 10 mg sorafenib (transarterial embolization with sorafenib [TAE-S] group). Liquid chromatography tandem mass spectrometry was used to measure sorafenib concentration in peripheral blood and tissue. Hepatic enzymes, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor 1α (HIF-1α) were measured at 0, 24, and 72 hours after treatment. Histopathologic examination was performed to evaluate extent of tumor necrosis and normal parenchymal damage. RESULTS: Serum sorafenib concentration peaked at 2 hours after treatment. The mean tissue concentration was 406.8 times greater than the serum concentration. Aspartate aminotransferase and alanine aminotransferase levels were significantly elevated in the TAE-S group at 24 hours after treatment. Serum VEGF and HIF-1α concentrations were not significantly different between the TAE-L and TAE-S groups. Hepatic parenchymal damage was more severe in the TAE-S group. Mean fraction of tumor necrosis after treatment was significantly greater in the TAE-S group. CONCLUSIONS: Transarterial chemoembolization using sorafenib resulted in a high intrahepatic concentration of sorafenib. The degree of tumor necrosis was significantly greater in the TAE-S group compared with the TAE-L group, but more severe toxicity of normal liver tissue also occurred.

A Single Meal Containing Raw, Crushed Garlic Influences Expression of Immunity- and Cancer-Related Genes in Whole Blood of Humans.

BACKGROUND: Preclinical and epidemiologic studies suggest that garlic intake is inversely associated with the progression of cancer and cardiovascular disease. OBJECTIVE: We designed a study to probe the mechanisms of garlic action in humans. METHODS: We conducted a randomized crossover feeding trial in which 17 volunteers consumed a garlic-containing meal (100 g white bread, 15 g butter, and 5 g raw, crushed garlic) or a garlic-free control meal (100 g white bread and 15 g butter) after 10 d of consuming a controlled, garlic-free diet. Blood was collected before and 3 h after test meal consumption for gene expression analysis in whole blood. Illumina BeadArray was used to screen for genes of interest, followed by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on selected genes. To augment human study findings, Mono Mac 6 cells were treated with a purified garlic extract (0.5 µL/mL), and mRNA was measured by qRT-PCR at 0, 3, 6, and 24 h. RESULTS: The following 7 genes were found to be upregulated by garlic intake: aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT), hypoxia-inducible factor 1α (HIF1A), proto-oncogene c-Jun (JUN), nuclear factor of activated T cells (NFAT) activating protein with immunoreceptor tyrosine-based activation motif 1 (NFAM1), oncostatin M (OSM), and V-rel avian reticuloendotheliosis viral oncogene homolog (REL). Fold-increases in mRNA transcripts ranged from 1.6 (HIF1A) to 3.0 (NFAM1) (P < 0.05). The mRNA levels of 5 of the 7 genes that were upregulated in the human trial were also upregulated in cell culture at 3 and 6 h: AHR, HIF1A, JUN, OSM, and REL. Fold-increases in mRNA transcripts in cell culture ranged from 1.7 (HIF1A) to 12.1 (JUN) (P < 0.01). OSM protein was measured by ELISA and was significantly higher than the control at 3, 6, and 24 h (24 h: 19.5 ± 1.4 and 74.8 ± 1.4 pg/mL for control and garlic, respectively). OSM is a pleiotropic cytokine that inhibits several tumor cell lines in culture. CONCLUSION: These data indicate that the bioactivity of garlic is multifaceted and includes activation of genes related to immunity, apoptosis, and xenobiotic metabolism in humans and Mono Mac 6 cells. This trial is registered at clinicaltrials.gov as NCT01293591.

α-tocopherol supplementation prevents lead acetate and hypoxia-induced hepatic dysfunction.

OBJECTIVE: Lead (Pb) is a long-known poison of environment and industrial origin. Its prolonged exposure affects cellular material and alters cellular genetics and produces oxidative damages. In this study, we investigated the exposure of chronic sustained hypoxia or lead acetate alone or in combination with or without supplementation of α-tocopherol on hepatic oxidative and nitrosative stress in rats. MATERIALS AND METHODS: The rats weighing 165 ± 5 g were exposed to chronic sustained hypoxia (10% oxygen) or lead acetate (25 mg/kg of body weight, intraperitoneally) alone or in combination with or without supplementation of α-tocopherol (10 mg/100 g b.wt, intramuscularly). The body weight of all the rats was recorded on the day 1 of the treatment and the day of sacrifice. Serum lipid profile was estimated by using a biochemical analyzer. Oxidant and enzymatic antioxidants status was evaluated by using spectrophotometer. Serum levels of hypoxia inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) were measured by using ELISA technique. Histopathological assessments of hepatic tissue were also done. RESULTS: Exposure of both lead and hypoxia showed decreased body weight, altered serum lipid profile, oxidant and enzymatic antioxidants status, serum HIF-1α and VEGF concentrations. Simultaneous α-tocopherol supplementation showed beneficial effects to all these alterations. Histopathological observations also showed hepatic degenerative changes after lead or hypoxia exposure either alone or in combination, but remarkable improvement has been noticed after α-tocopherol supplementation. CONCLUSION: Supplementation of α-tocopherol is beneficial to counter both lead acetate and hypoxia induced hepatic cytotoxicities possibly by reducing oxidative and nitrosative stress.

Bone marrow angiogenesis in patients presenting with differential Chinese medicine syndrome: correlation with the clinico-pathological features of aplastic anemia.

OBJECTIVE: To explore differences in bone marrow angiogenesis seen in aplastic anemia (AA) patients presenting with differential Chinese medicine (CM) syndrome, and to correlate these differences with clinical pathology. METHODS: Thirty-five patients were enrolled, including 18 with "yang deficiency syndrome" and 17 with "yin deficiency syndrome." Bone marrow biopsies and serum were collected. Microvessel density (MVD) and positive expression of vascular endothelial-derived growth factor (VEGF) were detected by immunohistochemisty. Hypoxia inducible factor -1α (HIF-1α), and VEGF expression were assayed by enzyme-linked immunoabsorbent assay (ELISA), serum lactate dehydrogenase (LDH) was tested by enzyme method and liquid chip technology was used to detected the expression of interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. RESULTS: Counts for leukocytes, absolute neutrophils and platelets in "yin deficiency syndrome" were lower than those found in "yang deficiency syndrome" (P<0.05). MVD and VEGF expression, and the positive rate of CD34 and VEGF in bone marrow were lower in AA, especially in "yin deficiency syndrome" (P<0.01 or P<0.05). "Yin deficiency syndrome" displayed decreased VEGF and LDH expression, and enhanced expression of HIF-1α as compared to "yang deficiency syndrome" (P<0.05). Levels of IL-4 and IL-6 were higher in AA (P<0.01), but IL-10 was decreased (P<0.05). High TNF-α expression was seen in "yang deficiency syndrome" and IFN-γ expression was decreased in "yin deficiency syndrome" as compared with normals (P <0.01 and P<0.05, respectively). CONCLUSION: AA patients have lower MVD than normals, especially in "yin deficiency syndrome." MVD might differentially correlate to disease severity, and could be dependent on bone marrow or serum VEGF expression and LDH. Additionally, IL-2, IL-10, IL-4 and IFN-γ were negatively associated while IL-6 and TNF-α were positively associated with MVD.

[Regulation of erythropoiesis in patients with suppressed hematopoiesis during mountain climatic treatment].

AIM: to estimate the regulation of erythropoiesis and the coagulation system in patients with suppressed hematopoiesis in a mountain hospital (3200 m above sea level). SUBJECTS AND METHODS: The investigation included 12 patients with aplastic anemia (AA) and 10 with idiopathic thrombocytopenic purpura (ITP). Blood was received at a Bishkek hospital, then on days 20 and 40 of stay in the mountains. The authors studied erythropoietin (EPO) by enzyme immunoassay (Protein Contour kit, Russia), serum ferritin (SF) by immunoradioassay (Immunotech kit, Czech Republic), hypoxia-inducible factor-1alpha (HIF-1alpha), homocysteine (HC), hepcidin, endothelin (ET), and thrombomodulin (TM) by sandwich enzyme immunoassay, by applying monospecific antisera and monoclonal antibodies against relevant antigens (IDG Int Inc, USA). RESULTS: On staying in the mountains, there was a gradual increase in the content of hemoglobin in patients with AA and ITP. On day 40, in keeping with higher hemoglobin (Hb) levels, both groups showed a decrease in HIF-1alpha concentrations to the normal values (from 8.2 to 4.5 pg/ml). Due to the anemic syndrome, baseline EPO was increased by 5-7 times in the patients from both groups. On days 20-40, the content of EPO showed a 1.3-2.5-fold increase. In AA, HC was almost 3 times greater than the normal values; in ITP, it was 1.5-fold increased. On day 20 and during the patients'stay in the mountains, the level of HC remained in the normal range in both groups. CONCLUSION: Hypoxic hypoxia positively affects a number of hematological parameters, by normalizing erythropoiesis (Hb, EPO, and HIF-1alpha), iron metabolism (SF), and the coagulation system (HC, ET, and TM).

Glycine pretreatment ameliorates liver injury after partial hepatectomy in the rat.

BACKGROUND: Living donor liver transplantation subjects the donor to a major hepatectomy. Pharmacological or nutritive protection of the liver during the procedure is desirable to ensure that the remnant is able to maintain sufficient function. The aim of our study was to analyze the effects of pretreatments with alpha-tocopherol (vitamin E), the flavonoid silibinin and/or the amino acid L-glycine on the donor in a rat model. METHODS: Male Wistar rats were pretreated with L-glycine (5% in chow, 5 days), alpha-tocopherol (100 mg/kg body weight by gavage, 3 days) and/or silibinin (100 mg/kg body weight, i.p., 5 days). Thereafter, 90% partial hepatectomy was performed without portal vein clamping. RESULTS: Glycine pretreatment markedly decreased transaminase release (AST, 12 hr: glycine 1292 +/- 192 U/L, control 2311 +/- 556 U/L, p < .05; ALT, 12 hr: glycine 1013 +/- 278 U/L, control 2038 +/- 500 U/L, p < .05), serum ALP activity and serum bilirubin levels (p < .05). Prothrombin time was reduced, and histologically, liver injury was also decreased in the glycine group. Silibinin pretreatment was less advantageous and pretreatment with alpha-tocopherol at this very high dose showed some adverse effects. Combined, i.e., triple pretreatment was less advantageous than glycine alone. Liver resection induced HIF-1alpha accumulation and HIF-1alpha accumulation was also decreased by glycine pretreatment. CONCLUSION: The decrease of liver injury and improvement of liver function after pretreatment with glycine suggests that glycine pretreatment might be beneficial for living liver donors as well as for patients subjected to partial hepatectomy for other reasons.